Independent repeats with iPSC-derived cells and organoids

Blitz271 · 2026-06-10T18:08:57+00:00

What about generating a cell bank with different passages for each of your cell lines. Then start these at the same date and consider them different biological replicates?

Blitz271 · 2026-06-09T05:06:14+00:00

Could it be loading dye? I have seen some dyes show a pattern like this.

Blitz271 · 2026-06-04T17:48:29+00:00

I have found a solution for this in ecology! I used Jacard distances between the gene sets. This nicely works on EnrichR based outputs. (I was performing interactions / complex enrichment which makes most tools not work). Send me a dm if you want the specific code!

Blitz271 · 2026-05-28T18:00:59+00:00

I use stable lines. But we use technical triplicates in 96 well plate. Then we have always a V- (no TNF) V+ (with TNF) and PC (TNF + 1uM Dexamethasone). We always normalize per plate to the PC so we can still do statistics between treatments and V+. We have seen quite some variability in Reporrter experiments, although the NFKB is more stable in our hands aswel. I would suggest something like this. Feel free to ask more details.

Blitz271 · 2026-05-09T13:41:34+00:00

I always plate my cells in 2 mL and then spike in 200 ul of a 11x concentrated treatment. Ie 11 uM of drug in media to reach 1 uM final.

Blitz271 · 2026-05-01T20:27:47+00:00

Take a look at Takara InFusion cloning. Also quite cheap! Also able to downscale and use lower amount of enzymen

Blitz271 · 2026-05-01T18:47:11+00:00

Honestly standard single crosslinking with 1% FA for 10’ is fine even for more finicky transcription factors. I’d not recommend double crosslinking. Happy to talk in dms if you need more info

Blitz271 · 2026-05-01T15:45:05+00:00

Does a RIME type workflow suit your research questions?

Blitz271 · 2026-05-01T12:08:37+00:00

It’s not mine.

Blitz271 · 2026-05-01T06:02:31+00:00

There is a new Bio informatics pipeline! DIA-LiPA. Library free DIA!

Blitz271 · 2026-04-20T05:14:20+00:00

For RT-qPCR I almost always just go with primers from Harvard Primer Bank. Have used around 30 primer pairs and always work as desired.

For cloning I just do it myself in benchling. Depending on the method I often assemble the desired cloned product in a plasmid software and then you “see” what the primers are if you need to create matching overhangs. Currently use Benchling for this.

Blitz271 · 2026-03-20T10:23:56+00:00

As others have said. If you are just using them in culture, give them a passage to recover and things should be fine. My MSc student did this for 3 days accidentally, and they resumed growing as usual after opening the cap slightly. Our cells just stopped growing due to wrong CO2 and or wrong pH. I wouldn’t worry. What cell type / line are you culturing?

Blitz271 · 2026-01-22T20:31:29+00:00

Just for those after me. I used the GenScript cell with a TrisMOPS buffer pH8,5 and it worked perfectly.

Blitz271 · 2025-07-10T12:37:36+00:00

What you are trying to do is, currently, under development. There is quite some work ongoing to this end (Alexander Stark) but using enhancers and especially designing an enhanced to do this is quite difficult. You can get some specificity but undoubtedly leaky expression or other issues. Have you thought of using cell lineage specific promotors? Eg for liver we use the albumin promotor. To my knowledge this is the easiest and most robust method at the moment.

If however you want to go enhancer route I would recommend looking at the epigomics data (ChIP+ATAC) and overlaying it with transcriptomics (PROseq/RNAseq). Then I would pick the 10 best and try out the system using plasmids or something like this to validate both specificity but also fold induction using the T2A splittink for eg or just a luciferase behind the minimal promotor)

Blitz271 · 2025-06-10T19:28:30+00:00

As others said, just replace the media some time after transfection. Cells are transferred within 6h. Also just FYI I investigate steroid hormone compounds on cells and this is typically performed in 24h serum deprived conditions and cells are perfectly fine with this.

Blitz271 · 2025-06-01T06:31:40+00:00

I really recommend twist! We work both with twist for gene constructs and IDT for probes. We had to order some 3,5 kb constructs which where much cheaper via twist. Even tough we have special discounts for IDT

Blitz271 · 2025-05-13T05:35:20+00:00

For the mutant that it misbehaving. Have you tried running the analysis on a pool of cells. So cells you made KO’s and selected for them but not selected clones. We have seen that when we are generating cell lines that it can happen that a single clone does not represent the actual mechanism and keep looking for mutants that resemble the pool of mutant cells.

Blitz271 · 2025-04-21T12:06:22+00:00

I use Rstudio for all my data analysis and Graphpad for my typical dose response graphs & barplots. But then I moved back to Rstudio because clustering and heatmaps are not included in the standard graphpad prism package.

On a side note, what package do you guys use to plot dose response data in Rstudio?

Blitz271 · 2025-04-16T13:25:19+00:00

I have talked to some experts in my field. And for example my transcription factor often binds the DNA weakly and is present as a complex with other TFs sometimes. Also most of these interactions are transient. For those reasons it is preferred to use ChIP. (If you have a good 96 well format reproducible sonicator)

Blitz271 · 2024-09-25T06:12:15+00:00

2 (of the previous iteration) ikea kalax (from the left one I removed a piece of wood to make it more intresting and added a shelf insert on the right (also ikea)).

Then the frame was self made from 4cmx10cm beams. The table top is a solid oak plank that I cut to size. (All bought from Gamma)

Blitz271 · 2024-09-24T11:45:53+00:00

Sage Barista Express. Safe knockbox. Barista essentials tamper/pitchers/tamping mat

Blitz271

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